Prediction of the impact of processing critical conditions for Listeria monocytogenes growth in artisanal dry-fermented sausages (salami) through a growth/no growth model applicable to time-dependent conditions

L. monocytogenes data from artificially contaminated salami batter samples and from published data were used to evaluate a newly-developed, time-dependent probability parameter (P_t), which was then incorporated in the existing and already validated simplified modelling approach (SMA), taking into account time in assessing the pathogen growth probability. The SMA was further expanded to quantify the impact of each preservative factor (f) to growth probability. The modified SMA (mSMA) was used to evaluate those environmental conditions that could decrease the safety status of artisanal salami. The dependence of the probability of growth, and of the growth of L. monocytogenes on temperature, pH, a_w, lactic acid and indigenous lactic acid bacteria of salami was predicted and the impact of each factor was quantified under dynamically changing conditions. Characteristics were measured in 5 salami batches and for two of them the predicted final population size was higher than the European Commission (EC) tolerated level for L. monocytogenes (100 CFU g⁻¹). Applying the mSMA to potential scenarios that included the contribution of starter cultures, the onset of Listeria inhibition was predicted earlier, preventing the pathogen to exceed its critical limit. Given its prediction capability, the mSMA is expected to assist small-sized enterprise users in the effective management of preventive and corrective measures in salami processing.     

Influence of high gas production during thermophilic anaerobic digestion in pilot-scale and lab-scale reactors on survival of the thermotolerant pathogens Clostridium perfringens and Campylobacter jejuni in piggery wastewater

Safe reuse of animal wastes to capture energy and nutrients, through anaerobic digestion processes, is becoming an increasingly desirable solution to environmental pollution. Pathogen decay is the most important safety consideration and is in general, improved at elevated temperatures and longer hydraulic residence times. During routine sampling to assess pathogen decay in thermophilic digestion, an inversely proportional relationship between levels of Clostridium perfringens and gas production was observed. Further samples were collected from pilot-scale, bench-scale thermophilic reactors and batch scale vials to assess whether gas production (predominantly methane) could be a useful indicator of decay of the thermotolerant pathogens C. perfringens and Campylobacter jejuni. Pathogen levels did appear to be lower where gas production and levels of methanogens were higher. This was evident at each operating temperature (50, 57, 65 °C) in the pilot-scale thermophilic digesters, although higher temperatures also reduced the numbers of pathogens detected. When methane production was higher, either when feed rate was increased, or pH was lowered from 8.2 (piggery wastewater) to 6.5, lower numbers of pathogens were detected. Although a number of related factors are known to influence the amount and rate of methane production, it may be a useful indicator of the removal of the pathogens C. perfringens and C. jejuni.     

Model of pathogen transmission between livestock and white-tailed deer in fragmented agricultural and forest landscapes

The study summarizes the current knowledge on infection and recovery of white-tailed deer and cattle, and integrates this knowledge into the Soil and Water Assessment Tool (SWAT) via a new add-on module SIR (Susceptible - Infected - Recovered) for predicting pathogen transmission between livestock and deer. New processes modeled by the SWAT-SIR model include: (a) seasonal changes in deer population and habitat; (b) resource selection and seasonal changes in foliage consumption by deer; (c) ingestion of pathogens with water, foliage and via grooming soiled hide by deer and grazing cattle; (d) infection and recovery of deer and co-grazing cattle; (e) pathogen shedding by infected animals; (f) survival of pathogens in manure; (g) kinetic release of pathogens from applied manure and fecal material. The model output is linked with ARC-GIS to allow spatial and temporal analysis of pathogen distribution across the watershed for specific land use, weather and management scenarios.     

Effects of plant extracts on microbial growth, color change, and lipid oxidation in cooked beef

The effects of butylated hydroxyanisole/butylated hydroxytoluene (BHA/BHT), grape seed extract (ActiVin), pine bark extract (Pycnogenol), and oleoresin rosemary (Herbalox) on microbial growth, color change, and lipid oxidation were investigated in cooked ground beef. When compared to the control, 1.0% ActiVin and Pycnogenol) effectively reduced the numbers of Escherichia coli O157:H7 and Salmonella Typhimurium, and retarded the growth of Listeria monocytogenes and Aeromonas hydrophila. Pycnogenol resulted in reductions of 1.7, 2.0, 0.8, and 0.4 log CFU/g, respectively, in numbers of E. coli O157:H7, L. monocytogenes, S. Typhimurium, and A. hydrophila, respectively, after 9 days of refrigerated storage. The color of cooked beef treated with ActiVin was less light (L*), more red (a*), and less yellow (b*) than those treated with BHA/BHT, Pycnogenol, and Herbalox. ActiVin and Pycnogenol effectively retained the redness in cooked beef during storage. The control showed significantly higher thiobarbituric acid-reactive substances (TBARS) and hexanal content over storage. BHA/BHT, ActiVin, Pycnogenol, and Herbalox retarded the formation of TBARS by 75%, 92%, 94%, and 92%, respectively, after 9 days, and significantly lowered the hexanal content throughout the storage period. Results of this work show that ActiVin and Pycnogenol are promising additives for maintaining the quality and safety of cooked beef.     

Roles of singlet oxygen and triplet excited state of dissolved organic matter formed by different organic matters in bacteriophage MS2 inactivation

Inactivation of bacteriophage MS2 by reactive oxygen species (ROS) and triplet excited state of dissolved organic matter (³DOM*) produced by irradiation of natural and synthetic sensitizers with simulated sunlight of wavelengths greater than 320 nm was investigated. Natural sensitizers included purified DOM isolates obtained from wastewater and river waters, and water samples collected from Singapore River, Stamford Canal, and Marina Bay Reservoir in Singapore. Linear correlations were found between MS2 inactivation rate constants (k_obs) and the photo-induced reaction rate constants of 2,4,6-trimethylphenol (TMP), a probe compound shown to react mainly with ³DOM*. Linear correlations between MS2 k_obs and singlet oxygen (¹O₂) concentrations were also found for both purified DOM isolates and natural water samples. These correlations, along with data from quenching experiments and experiments with synthetic sensitizers, Rose Bengal (RB), 3′-methoxyacetophenone (3′-MAP), and nitrite (₂NO⁻)$\left({{\text{NO}}_2}^{-}\right)$, suggest that ¹O₂, ³DOM*, and hydroxyl radicals (^•OH) could inactivate bacteriophage MS2. Linear correlations between MS2 k_obs and Specific Ultraviolet Absorption determined at 254 nm (SUVA₂₅₄) were also found for both purified DOM isolates and natural samples. These results suggest the potential use of TMP as a chemical probe and SUVA₂₅₄ as an indicator for virus inactivation in natural and purified DOM water samples.     

Development of a DNA microarray for detection and identification of Legionella pneumophila and ten other pathogens in drinking water

The safety and accessibility of drinking water are major concerns throughout the world. Consumption of water contaminated with infectious agents, toxic chemicals or radiological hazards represents a significant health risk and is strongly associated with mortality. Therefore, we have developed an oligonucleotide-based microarray using the sequences of 16S-23S rDNA internal transcribed spacer regions (ITS) and the gyrase subunit B gene (gyrB) found in the most prevalent and devastating waterborne pathogenic agents. This new diagnostic contains 26 specific probes and can simultaneously detect Aeromonas hydrophila, Klebsiella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa, Salmonella spp., Shigella spp., Staphylococcus aureus, Vibrio choleraeo, Vibrio parahaemolyticus, Yersinia enterocolitica and Leptospira interrogans. Testing was carried out against a total of 218 bacterial strains, including 53 representative strains, 103 clinical isolates and 62 strains of other bacterial species belonging to 10 genera and 48 species. The results were specific and reproducible, with a detection sensitivity of 0.1 ng DNA or 10⁴ CFU/ml achieved for pure cultures of each target organism. The diluted cultures and real drinking water samples were tested by the microarray with 100% accuracy. This novel diagnostic method is superior in time- and labor-efficiency to conventional bacterial culture and antiserum agglutination, and can be readily applied to epidemiological surveillance and other food safety applications.     

Elimination by ozone of Shigella sonnei in shredded lettuce and water

Several outbreaks of shigellosis have been attributed to the consumption of contaminated fresh-cut vegetables. The minimal processing of these products make it difficult to ensure that fresh produce is safe for consumer. Chlorine-based agents have been often used to sanitize produce and reduce microbial populations in water applied during processing operations. However, the limited efficacy of chlorine-based agents and the production of chlorinated organic compounds with potential carcinogenic action have created the need to investigate the effectiveness of new decontamination techniques. In this study, the ability of ozone to inactivate S. sonnei inoculated on shredded lettuce and in water was evaluated. Furthermore, several disinfection kinetic models were considered to predict S. sonnei inactivation with ozone. Treatments with ozone (1.6 and 2.2 ppm) for 1 min decreased S. sonnei population in water by 3.7 and 5.6 log cfu mL(-1), respectively. Additionally, it was found that S. sonnei growth in nutrient broth was affected by ozone treatments. After 5.4 ppm ozone dose, lag-phases were longer for injured cells recovered at 10 degrees C than 37 degrees C. Furthermore, treated cells recovered in nutrient broth at 10 degrees C were unable to grow after 16.5 ppm ozone dose. Finally, after 5 min, S. sonnei counts were reduced by 0.9 and 1.4 log units in those shredded lettuce samples washed with 2 ppm of ozonated water with or without UV-C activation, respectively. In addition, S. sonnei counts were reduced by 1.8 log units in lettuce treated with 5 ppm for 5 min. Therefore, ozone can be an alternative treatment to chlorine for disinfection of wash water and for reduction of microbial population on fresh produce due to it decomposes to nontoxic products.     

pilF polymorphism-based real-time PCR to distinguish Vibrio vulnificus strains of human health relevance

The Gram-negative bacterium Vibrio vulnificus is a common inhabitant of estuarine environments. Globally, V. vulnificus is a significant foodborne pathogen capable of causing necrotizing wound infections and primary septicemia, and is a leading cause of seafood-related mortality. Unfortunately, molecular methods for the detection and enumeration of pathogenic V. vulnificus are hampered by the genetically diverse nature of this pathogen, the range of different biotypes capable of infecting humans and aquatic animals, and the fact that V. vulnificus contains pathogenic as well as non-pathogenic variants. Here we report an alternative approach utilizing the development of a real-time PCR assay for the detection of pathogenic V. vulnificus strains based on a polymorphism in pilF, a gene previously indicated to be associated with human pathogenicity. Compared to human serum reactivity, the real-time PCR assay successfully detected pathogenic strains in 46 out of 47 analysed V. vulnificus isolates (97.9%). The method is also rapid, sensitive, and more importantly can be reliably utilised on biotype 2 and 3 strains, unlike other current methods for V. vulnificus virulence differentiation.     

Monitoring bacterial indicators and pathogens in cattle feedlot waste by real-time PCR

Quantitative microbial health risk assessment requires accurate enumeration of pathogens in hazard-containing matrices as part of the risk characterization process. As part of a risk management-oriented study of cattle feedlot waste contaminants, we investigated the utility of quantitative real-time PCR (qPCR) for surveying the microbial constituents of different faecal wastes. The abundance of Escherichia coli and enterococci were first estimated in five cattle feedlot waste types from five localities. Bacteria were quantified using two culture methods and compared to the number of genome copies detected by qPCR targeted at E. coli and Enterococcus faecalis. Bacterial numbers detected in the different wastes (fresh faeces, pen manure, aged manure, composted manure, carcass manure compost) ranged from 10−⁷ to 10² g⁻¹ (dry weight). Both indicator groups were detected by qPCR with a comparable sensitivity to culture methods across this range. qPCR measurements of E. coli and E. faecalis correlated well with MPN and spread plate data. As a second comparison, we inoculated green fluorescent protein (GFP) labeled reference bacteria into manure samples. GFP labeled E. coli and Listeria monocytogenes were detected by qPCR in concentrations corresponding to between 18% and 71% of the initial bacterial numbers, compared to only 2.5-16% by plating. Our results supported our selection of qPCR as a fast, accurate and reliable system for surveying the presence and abundance of pathogens in cattle waste.